Center for Veterinary Biologics SAM 500.06

Testing Protocol Page 1 of 11

United States Department of Agriculture 

Center for Veterinary Biologics

Testing Protocol 

SAM 500

Supplemental Assay Method for the Determination of Phenol in Veterinary 

Biologics (pullorum antigen, Mycoplasma synoviae antigen, and Mycoplasma 

gallisepticum antigen)

Date: June 16, 2022 

Number: SAM 500.06 

Supersedes: SAM 500.05, February 5, 2016 

Standard Requirement: 9 CFR 113.407-408 

Contact: Email: Methodsrequest.notification@usda.gov 

Phone: Center for Veterinary Biologics, 515-337-6100 

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844 

Ames, IA 50010 

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product 

by USDA and does not imply its approval to the exclusion of other products that may be suitable.

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Testing Protocol

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Supplemental Assay Method for the Determination of Phenol in Veterinary Biologics (pullorum antigen,

Mycoplasma synoviae antigen, and Mycoplasma gallisepticum antigen)

Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents 

3.4 Preparation of the sample 

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions

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Testing Protocol

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Supplemental Assay Method for the Determination of Phenol in Veterinary Biologics (pullorum antigen,

Mycoplasma synoviae antigen, and Mycoplasma gallisepticum antigen)

1. Introduction

This Supplemental Assay Method (SAM) describes the procedures for determination of phenol 

in the following veterinary biologics: pullorum tube antigen and avian mycoplasma antigens; 

Mycoplasma synoviae and Mycoplasma gallisepticum; as prescribed in title 9, Code of Federal 

Regulations (9 CFR), parts 113.407 and 113.408, respectively. 

Phenol concentration is determined by a direct titration with a standardized bromide-bromate 

solution. 

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below. 

2.1.1 Balance, analytical, capable of measuring 0.0001 g 

2.1.2 Balance, top loading, capable of measuring 0.01 g 

2.1.3 Volumetric pipettes, Class A, 5-mL

2.1.4 Volumetric flasks with barrel head glass stopper, Class A, 500-mL and 

1-L 

2.1.5 Burets with PTFE stopcocks, precision bore, Class A, 25- and 50-mL

2.1.6 Graduated cylinders, Class A, 10-, 50-, 100-, 250-, 500-, and 1,000-mL

2.1.7 Glass-stoppered Erlenmeyer flasks, 250-mL

2.1.8 Heating/stirring plate with stirring bars

2.1.9 Fast filter paper, Whatman No. 1 

2.1.10 Weigh boats, or equivalent 

2.1.11 Timers, 30 seconds to 1 minute

2.1.12 Dropper, i.e., transfer pipette, Pasteur pipette, dropper bottle

2.1.13 Pipettor and tips to accurately dispense 100- to 1000-μL

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Testing Protocol

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Supplemental Assay Method for the Determination of Phenol in Veterinary Biologics (pullorum antigen,

Mycoplasma synoviae antigen, and Mycoplasma gallisepticum antigen)

2.1.14 Stir plate

2.1.15 Stir bars

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

chemicals are reagent grade, unless specified.

2.2.1 Hydrochloric acid (HCl), CAS# 7647-01-0, Assay: 36.5-38.0% 

2.2.2 Water (H2O), Purity: distilled, demineralized, reverse osmosis or

equivalent. 

2.2.3 Methyl orange, CAS#547-58-0, Purity: 98.0% 

2.2.4 Silicotungstic acid hydrate (H4[Si(W3O10)4]*26H2O), CAS# 12027-43-9,

Purity: 99.0%, store at 4°C.

2.2.5 Sulfuric acid (H2SO4), CAS# 7664-93-9, Purity: Minimum 95.0%,

Maximum 98.0% 

2.2.6 Arsenic trioxide, anhydrous (As2O3), CAS# 1327-53-3, Purity: 99.9%

2.2.7 Sodium hydroxide (NaOH), CAS# 1310-73-2, Purity: 98.5% 

2.2.8 Phenol (C6H6O), CAS#108-95-2, Purity: ≥ 99.0%

This can be a purchased NIST standard and diluted, if necessary, to the 

appropriate level.

2.2.9 Sodium bicarbonate (NaHCO3), CAS#144-55-8, Purity: 99.9%

2.2.10 Potassium bromate (KBrO3), CAS#7758-01-2, Purity: 98.5%

2.2.11 Potassium bromide (KBr), CAS#7758-02-3, Purity: 99.0% 

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel must have experience or training in this protocol. This includes 

working knowledge of the use of general laboratory equipment, glassware and chemical 

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Supplemental Assay Method for the Determination of Phenol in Veterinary Biologics (pullorum antigen,

Mycoplasma synoviae antigen, and Mycoplasma gallisepticum antigen)

safety; and specific training in the operation of the laboratory equipment and reagents 

listed in Section 2. 

Analysts performing this procedure should first conduct 2 trial runs using controls and 

standards and obtain results within acceptable limits.

3.2 Preparation of equipment/instrumentation

All equipment must be operated according to manufacturers’ recommendations and 

monitored in compliance with applicable standard operating policies/procedures. 

Prime the buret by rinsing with test fluid.

3.3 Preparation of reagents

Reagents are stable for 6 months from date of preparation and stored at room 

temperature, unless otherwise noted. Prepare reagents in volumes appropriate to demand 

to minimize waste due to expiration.

Glassware used for preparation of reagents must meet ASTM requirements; 

measurements are based on the measurements of uncertainty outlined in those 

requirements.

In the following steps the acronym QS is used. It is defined as quantity sufficient; as 

much as is sufficient.

3.3.1 Standard, 0.25% phenol: Dissolve 2.50 ± 0.01g phenol in approximately 

500 mL water in a 1L volumetric flask; QS to 1L with water.

3.3.2 Control Sample:

Either (1) a pool of PPD tuberculin products with established protein and phenol 

values as tested by Pathobiology Laboratory-Chemistry and Analytical Services 

(PL-CAS); or (2) a product produced for use as a control sample and tested by 

PL-CAS.

(1) Combine any sample volumes remaining after test completion in a 

pool. Record all identifying information, CAS phenol result, and 

expiration date for each sample. Control sample phenol concentration is 

the mean of CAS results for all samples included in the pool. 

(2) Obtain a product produced for use as a control sample. Analyze the 

product a total of three times, mean of these trials must be 0.50 ± 0.04 % 

phenol. Expiration date is as indicated on product. Store at 4°± 10° C.

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Supplemental Assay Method for the Determination of Phenol in Veterinary Biologics (pullorum antigen,

Mycoplasma synoviae antigen, and Mycoplasma gallisepticum antigen)

3.3.3 20% Hydrochloric Acid (HCl): In a 1-L volumetric flask, slowly add 

200 mL hydrochloric acid to 600 mL water; QS to 1 L with water.

3.3.4 0.1% Methyl Orange: Dissolve 0.1 ± 0.01 g methyl orange in 100 mL 

water. Filter if necessary.

3.3.5 Silicotungstic acid solution (SAS): Dissolve 60.00 ± 0.5 g silicotungstic 

acid hydrate in 400 mL water in a 500-mL volumetric flask. Add 50 mL sulfuric 

acid. When cool, QS to 500 mL with water.

3.3.6 Clarifying solution (CS): Add 50 mL SAS and 125 mL 20% 

hydrochloric acid to 325 mL water. Prepare fresh prior to each test.

3.3.7 "Acid solution" for As2O3 standardization solution: Add 110 mL

hydrochloric acid and 2.5 mL 0.1% methyl orange to 100 mL water.

3.3.8 0.050 N Arsenic trioxide (As2O3): CAUTION!! Arsenic trioxide is

extremely toxic. Avoid contact; handle in fume hood using gloves, mask, and 

goggles. Consult the Safety Data Sheet for specific handling instructions before 

proceeding. Dissolve 2.4730 ± 0.001 g anhydrous arsenic trioxide in 25 mL hot 1 

N sodium hydroxide in a 1-L volumetric flask. Neutralize solution with 25 mL 1 

N sulfuric acid. When cool, QS to 1 L with water.

3.3.9 1 N Sodium hydroxide: Dissolve 4.00 ± 0.01 g of sodium hydroxide in 

60 mL water in a 100-mL volumetric flask; QS to 100 mL with water.

3.3.10 1 N Sulfuric acid: In a 100-mL volumetric flask, slowly add 4.904 mL 

sulfuric acid to 60 mL water; QS to 100 mL with water.

3.3.11 Test fluid (TF): Dissolve 0.30 ± 0.01 g sodium bicarbonate, 1.67 ± 

0.01 g potassium bromate, and 15.00 ± 0.01 g potassium bromide in water and QS 

to 1 L with water. CRITICAL CONTROL POINT: The test fluid must be 

standardized as described in 3.3.11(1) prior to use.

1. Standardization

a. Prepare standardization solution: Add 25 mL 0.050 N arsenic 

trioxide to 10 mL “Acid Solution.” 

b. Confirm standardization solution by titrating with previous lot of 

TF. It should take 21.3 mL test fluid (TF) to titrate the standardization 

solution. 

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Supplemental Assay Method for the Determination of Phenol in Veterinary Biologics (pullorum antigen,

Mycoplasma synoviae antigen, and Mycoplasma gallisepticum antigen)

c. Titrate standardization solution with new lot of TF. The 

required titration volume is 21.3 mL TF. A first time titration may 

require less than 21.3 mL TF, in which case, the TF volume must 

be adjusted by adding the correct volume of water to the TF, 

continue to Step 1d if this is the case. If the first time titration is

21.3 ± 0.1 mL TF, Step 2. 

d. Adjust the TF volume. For this step the calculations are 

shown, and an example is used to illustrate.

A = Starting volume of TF (mL) 

B = Titration volume of TF (mL) 

C = Volume of TF left (mL)

D = Required titration volume (21.3 mL) 

E = Adjusted volume of TF (mL) 

F = Volume of water to be added to volume of TF left to 

achieve the adjusted volume (mL) 

Example: Assume the starting volume of TF is 1000 mL and the titration volume 

is 20.5 mL. 

• A – B = C

Example: (1000 mL) - (20.5 mL) = 979.5 mL

• (C) (D) = E

(B) 

Example: 

(979.5 mL) (21.3 mL) = 1017.7 mL

(20.5 mL) 

• E – C = F

Example: (1017.7 mL) - (979.5 mL) = 38.2 mL 

• Add the calculated volume of water (F) to the existing TF and put any 

TF remaining in the buret back into flask. Continue to Step 2.

2. Repeat Step 1c until three consecutive trials produce an average titration 

volume of 21.3 mL.

3.4 Preparation of the sample 

3.4.1 Receipt

Complete sample receipt as described by standard operating procedures. 

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Testing Protocol

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Supplemental Assay Method for the Determination of Phenol in Veterinary Biologics (pullorum antigen,

Mycoplasma synoviae antigen, and Mycoplasma gallisepticum antigen)

3.4.2 Preparation

Licensed or prelicense biologics products are generally received in sealed serum 

bottles and stored at 4°± 10°C prior to testing. Before testing, allow sample vials 

and reagents to come to room temperature.

4. Performance of the Test

4.1 Pullorum tube antigen 

Analyze the control pool and phenol standard each time testing is performed. Analyze 

control and standard in duplicate or triplicate, and samples in triplicate.

4.1.1 Add 5 mL sample and 50 mL 20% HCl to a 250-mL glass-stoppered flask. 

Shake until the solution decolorizes (final appearance will be white-cloudy,

typically takes 2 to 3 minutes). Add 50 mL H2O and mix. Filter through filter

paper and collect 50 mL of filtrate.

4.1.2 Transfer 50 mL of filtrate to another 250-mL glass-stoppered flask. Add a 

stir bar and place flask on stir plate with buret directly above. Add 1 drop 0.1% 

methyl orange (indicator), stir for a few seconds. Observe the color as pink. 

An acceptable alternative to using a stir plate and stir bar would be shaking the 

flask.

4.1.3 Titrate with 2 mL test fluid (TF), stir or shake for a few seconds. Observe 

the color; if pink, repeat. If colorless, go to Section 4.1.4. 

4.1.4 Stir or shake 30 seconds. Add 1 drop indicator, stir for a few seconds. 

Observe the color. If colorless for ≤ 10 seconds or if pink, titrate with 1 mL TF, 

and repeat. If colorless for ≥ 10 seconds, go to Section 4.1.5. 

4.1.5 Stir or shake 1 minute. Add 1 drop indicator, stir for a few seconds. 

Observe the color. If pink for ≥ 10 seconds, titrate with 0.50 mL TF, and repeat. 

When colorless within 10 seconds, record total volume of TF as the endpoint of 

titration and use this volume for calculation of percent phenol. 

4.2 Mycoplasma gallisepticum and Mycoplasma synoviae antigen 

Analyze the control pool and phenol standard each time testing is performed. Analyze 

control and standard in duplicate or triplicate, and samples in triplicate.

4.2.1 Combine 5 mL sample and 100 mL clarifying solution (CS) to a 250-mL

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Supplemental Assay Method for the Determination of Phenol in Veterinary Biologics (pullorum antigen,

Mycoplasma synoviae antigen, and Mycoplasma gallisepticum antigen)

glass-stoppered flask. Shake 2 minutes. Filter through filter paper and collect 

50 mL of filtrate.

4.2.2 Transfer 50 mL of filtrate to another 250-mL glass-stoppered flask. Add a 

stir bar and place flask on stir plate with buret directly above. Add 1 drop 0.1% 

methyl orange (indicator), stir for a few seconds. Observe the color as pink. 

An acceptable alternative to using a stir plate and stir bar would be shaking the 

flask.

4.2.3 Titrate with 2 mL TF, stir or shake for a few seconds. Observe the color; 

if pink, repeat. If colorless, go to Section 4.2.4. 

4.2.4 Stir or shake 30 seconds. Add 1 drop indicator, stir for a few seconds. 

Observe the color. If colorless for ≤ 10 seconds or if pink, titrate with 1 mL TF, 

and repeat. If colorless for ≥ 10 seconds, go to Section 4.2.5. 

4.2.5 Stir or shake 1 minute. Add 1 drop indicator, stir for a few seconds. 

Observe the color. If pink for ≥ 10 seconds, titrate with 0.50 mL TF, and repeat. 

When colorless within 10 seconds, record total volume of TF as the endpoint of 

titration and use this volume for calculation of percent phenol. 

5. Interpretation of the Test Results

5.1 Pullorum tube antigen (report average of triplicates)

% Phenol = (volume of test fluid) x (0.04) 

Satisfactory Phenol Content according to 9 CFR 113.407: 0.55 ± 0.05%. 

5.2 Mycoplasma gallisepticum and Mycoplasma synoviae antigen (report average of 

triplicates)

% Phenol = (volume of test fluid) x (0.04) 

Satisfactory Phenol Content according to 9 CFR 113.408: 0.25 ± 0.05%. 

5.3 Controls 

Results for controls and standards must be within acceptable limits; otherwise repeat 

testing.

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Supplemental Assay Method for the Determination of Phenol in Veterinary Biologics (pullorum antigen,

Mycoplasma synoviae antigen, and Mycoplasma gallisepticum antigen)

6. Report of Test Results

Report results of the test(s) as described by standard operating procedures. 

7. References

7.1 Title 9, Code of Federal Regulations, parts 113.407-408, U.S. Government 

Printing Office, Washington, DC.

7.2 ASTM Standard E969, Standard Specification for Glass Volumetric (Transfer) 

Pipets

7.3 ASTM Standard E288, Standard Specification for Laboratory Glass Volumetric 

Flasks

7.4 ASTM Standard E694, Standard Specification for Laboratory Glass Volumetric 

Apparatus 

8. Summary of Revisions 

Version .06 

• Updated coversheet and contact information. 

Version .05 

• Updated Contact information.

• Includes option to purchase a NIST standard phenol. 

• Updated control sample information. 

Version .04 

• The Contact information has been updated. 

• 3.1: Personnel qualifications/training has been revised.

• 7: ASTM Standards E969, E288 and E694 have been added as references.

• All references to chemicals in procedural steps have been changed from the chemical 

formula to the chemical name

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Supplemental Assay Method for the Determination of Phenol in Veterinary Biologics (pullorum antigen,

Mycoplasma synoviae antigen, and Mycoplasma gallisepticum antigen)

• The entire document has been revised to reflect current practices.

Version .03 

• The document number has been changed from TCSAM0500 to SAM 500. 

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